PROJECT SUMMARY Heart rhythm is triggered and maintained by synchronized electrical impulses throughout the heart. The slow delayed rectifier potassium current (IKs) vitally contributes to proper repolarization of cardiac action potentials, and is thus essential for maintaining a healthy heart rhythm. The molecular correlate of IKs was identified to be an ion channel complex, formed by two integral membrane proteins: KCNQ1 and KCNE1. KCNQ1 (also known as Kv7.1 or KvLQT1) is the pore-forming subunit of the IKs channel complex. It belongs to the voltage-gated potassium channel superfamily. Expression of KCNQ1 alone generates a rapidly activating and inactivating delayed-rectifier potassium current, whose properties, however, do not match with those of the cardiac IKs. KCNQ1 must co-assemble with its ancillary subunit, KCNE1 to produce the IKs current. KCNE1 is a small single-transmembrane protein that profoundly modifies the biophysical properties of KCNQ1 by slowing activation and deactivation kinetics, by shifting the voltage-dependence of channel open probability, and by increasing the single channel conductance. Despite intensive studies on IKs in the past decade it remains largely unclear how KCNE1 modulates and alters the function of KCNQ1 at molecular level. On the other hand, due to its vital role in cardiac function, mutations in kcnq1 or kcne1 gene can lead to several cardiac diseases such as familial atrial fibrillation, long-QT syndromes, short-QT syndromes and even sudden death in infants. Yet, as a potential drug target, molecular determinants underlying how small molecules could potentially manipulate the function of KCNQ1/KCNE1 channel complexes are largely unknown. Here I propose to carry out systematic structure-based investigations on KCNQ1/KCNE1 by achieving three immediate goals: 1) Structural and biochemical characterization of the KCNQ1/KCNE1 channel complex; 2) High-throughput small molecule screen using a proteoliposome-based flux assay; 3) Structural and functional elucidation of the interaction between the KCNQ1/KCNE1 complex and small molecules. During the K99 mentored phase in Dr. Roderick MacKinnon's laboratory, I will carry out single particle cryo-EM study to determine the high-resolution structure of the IKs channel complex. At the same time, I will establish a proteoliposome-based flux assay, by which high-throughput small molecule screens can be carried out to search for compounds targeting the KCNQ1/KCNE1 channel complex. In the R00 independent phase, large-scale small molecule screens will be done, and promising hits will be characterized using both biochemical and biophysical methods such as cell- based patch clamp assays. Finally, molecular details underlying interactions between the IKs channel complex and small molecules will be investigated by biophysical and biochemical approaches. My research will unveil the molecular nature of IKs, provide a blueprint for structure-based drug design, and serve as a paradigm for studying ion channel modulation by single transmembrane accessory subunits and small molecules.